The hydrogen concentration, or the pH, is an extremely important parameter in biological and chemical/technical systems. Many chemical and biological reactions require an exact regulation of the pH for a proper course. For example, a complex natural process takes place for regulating the pH in human blood, which normally has a pH of approximately 7.4. Even changes of just a few tenths of a pH unit can be due to or cause serious disease states. Although a multiplicity of techniques has been developed to measure pH, these are generally based on either electrochemical or optical principles. A standard laboratory pH measurement device, for example comprises a standard electrode of known electric potential, a special glass electrode which changes electric potential as a function of the concentration of hydrogen ions in the medium into which it is being dipped and a potentiometer which measures the electric potential between the two electrodes, from which a numerical pH is determined. Methods of this type are not however very good for measurements in intact biological systems, as a measurement electrode needs to be inserted.
In the context of optical measurements, pH indicators are used, which pH indicators are dyes whose detectable optical characteristics, such as extinction (absorption) or fluorescence likewise change with the change of the pH. Thus, pH indicators indicate the current pH of the solution by means of their color shade and their color intensity. The greatest sensitivity of indicators to small changes of pH is present if the equilibrium constant (pKa) between the acidic and basic forms of the indicator is close to the pH of the medium to be investigated, generally a solution.
As a broad generalization, optical measurements of the pH are regarded as being inferior to the electrochemical techniques, mainly because factors different from the hydrogen ion concentration, such as temperature, ionic strength and protein concentration can influence the dyes and interfere with the pH measurements. Nevertheless, optical techniques have enormous advantages when costs and size play a role and especially when the pH should be measured in living cells and tissues, into which no measurement probe can be inserted. Measurements of pH-dependent emission intensity in individual cells with a single excitation wavelength suffer from inaccuracies which relate to dye concentration, photobleaching of the dye, thickness of the cell measured or path length. A solution of the problem of dye concentration consists in the determination of the ratio of the fluorescence intensity at a fixed wavelength with excitation at a pH-sensitive wavelength to fluorescence intensity at the same wavelength with excitation at a relatively pH-insensitive wavelength. This method is usually used in order to estimate the pH in the interior of cells with fluorescence derivatives and is suitable in practice for suspensions of cells and in homogeneous liquids, such as media in a research fluorometer or microscope.
An optical biosensor is described in DE 39 23 921 A1, in the case of which biosensor an enzyme which catalyses a chemical reaction is bonded to a pH indicator. The indicator can be a fluorescent dye, particularly one based on coumarin. A change of the pH during the chemical reaction can be determined due to measurable change of the fluorescence intensity of the dye. A culture medium for the detection and the determination of numbers of microorganisms is described in DE 600 13 613 T2, which culture medium comprises a pH indicator which is chemically bonded to a hydrophilic substance with a high molecular weight. Thus, a water-soluble pH indicator with ballast is provided, which at the same time, as a nutrient gel, supports the hydrated growth of the microorganisms. Growth detection is brought about by the detection of the change in fluorescence of the pH indicator, which can contain carboxyphenol red, in the culture medium. DE 101 52 994 A1 describes an optical method for the simultaneous determination of pH and dissolved oxygen. To this end, two fluorescent, for example, ruthenium-based, pH indicators are used in a common matrix in the medium.
A pH indicator based on fluorescent xanthine dyes is described in U.S. Pat. No. 4,945,171. Here, two emission maxima are measured during the excitation with just one wavelength with particular selectivity for the independent excitation of acid and base form at either a single or two wavelength(s) and the pH-dependent absorption or fluorescence excitation spectra resulting therefrom. A pH indicator based on fluorescent carbazine dyes and derivatives is described in DE 196 81 363 C2. Carbazine dyes exhibit greater fluorescence, greater stability, greater temperature sensitivity and greater Stokes shifts compared to xanthine dyes. Additionally, the carbazine dyes are better to immobilize on a solid support. A method for indicating the pH of a solution as medium is disclosed, in the case of which the pH indicator is added to the solution and brought into contact with light of a chosen wavelength, in order to excite the carbazine dye to fluorescence, the intensity of the fluorescence at two different wavelengths is measured and the ratio of the fluorescence intensities at the two chosen wavelengths is calculated and the ratio is correlated with a predetermined relationship for such ratios with the pH.
Further fluorescent dyes which are used for measuring pH are, for example, the dyes BCECF and DNP-160. Further molecular probes from the pH indicator family can, for example, be drawn from the following table (sorted by descending equilibrium constant pKa, tables taken from http://probes.invitrogen.com/handbook/tables/0361.html, as of 27.05.2007). The small size of the detectable pH range can be seen clearly in each case.
PRECURSORPHFLUOROPHORERANGETYPICAL MEASUREMENTSNARF indicators6.0-8.0Emission rate 580/640 nmHPTS (pyranine)7.0-8.0Excitation rate 450/405 nmBCECF6.5-7.5Excitation rate 490/440 nmFluoresceins and6.0-7.2Excitation rate 490/450 nmcarboxyfluoresceinsLysoSensor Green DND-1894.5-6.0Individual emission 520 nmOregon green dyes4.2-5.7Excitation rate 510/450 nmor 490/440 nmLysoSensor3.5-6.0Emission rate 450/510 nmyellow/blue DND-160
The bioactive natural marine substance ageladine A (chemical formula C10H7N5Br2) is a pyrrole-imidazole alkaloid, which can be isolated from sponges of the genus Agelas for example (cf. Publication I by M. Fujita et al.: “Ageladine A: An Antiangiogenetic Matrixmetalloproteinase Inhibitor from Marine Sponge Agelas nakamurai”, J. Am. Chem. Soc. 2003, 125, 15700 15701 and Supporting Information S.I. 1-15). Ageladine A can meanwhile also be completely synthesized (cf. Publication II by M. Meketa et al.: “Total Synthesis of Ageladine A, an Angiogenesis Inhibitor from the Marine Sponge Agelas nakamurai” Org. Lett. 2006, 8, 7, 14431446, Publication III BY S. Shengule et al.: “Concise Total Synthesis of the Marine Natural Product Ageladine A”, Org. Lett. 2006, 8, 18, 4083-4084 and Publication IV by M. Meketa et al.: “A New Total Synthesis of the Zinc Matrixmetalloproteinase Inhibitor Ageladine A Featuring a Biogenetically Patterned 67t-2-Azatriene Electrocyclization”, Org. Lett. 2007, 9, 5, 853-855). Thus, ageladine A is publicly available in an unlimited amount. In scientific investigations of ageladine A with measurements for the cellular action of natural marine substances, ageladine A exhibits a disruptive autofluorescence (cf. Publication V by U. Bickmeyer et al.: “Disturbance of Voltage Induced Cellular Calcium Entry by the Marine Pyrrole-Imidazole Alkaloids”, doi:10.1016/j.toxicon.2007.04.015). Following UV excitation, ageladine A has a pronounced fluorescence in the green range (see Publication I).
DE 10 2004 002 885 B4 describes a range of new bioactive compounds from the class of pyrrole alkaloids. In this case, marine sponges are a rich source of pyrrole alkaloids, a group of natural substances which stands out on account of its structural variety and interesting biological activities. Mention is also made of pyrrole-imidazole alkaloids obtained from various Caribbean sponge species of the genus Agelas, with a selective antihistaminic effect and Agelongin (structural formula 22) from Agelas longissima with an antiserotonergic effect on fundus preparations from rat stomachs. All bioactive compounds mentioned are used exclusively for medical purposes, particularly for combating neurodegenerative diseases.